Selective modulation of coupled ryanodine receptors during microdomain activation of calcium/calmodulin-dependent kinase ii in the dyadic cleft

Dries, E., Bito, V., Lenaerts, I., Antoons, G., Sipido, K. R. and MacQuaide, N. (2013) Selective modulation of coupled ryanodine receptors during microdomain activation of calcium/calmodulin-dependent kinase ii in the dyadic cleft. Circulation Research, 113(11), pp. 1242-1252. (doi: 10.1161/CIRCRESAHA.113.301896) (PMID:24081880)

Abstract

Rationale: In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Objective: Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. Methods and Results: We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca²⁺ transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K₅fluo-4 as Ca²⁺ indicator). Ca²⁺ sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca²⁺/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca²⁺/calmodulin-dependent kinase II blockers autocamtide-2–related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca²⁺/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca²⁺ transients, frequency-dependent increase of the rate of Ca²⁺ rise was seen in coupled RyR regions only and abolished by autocamtide-2–related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Conclusions: Coupled RyRs have a distinct modulation by Ca²⁺/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca²⁺/reactive oxygen species microdomain, and subject to modification with disease.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: MacQuaide, Dr Niall
• Authors: Dries, E., Bito, V., Lenaerts, I., Antoons, G., Sipido, K. R., and MacQuaide, N.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Circulation Research
• Publisher: American Heart Association
• ISSN: 0009-7330
• ISSN (Online): 1524-4571